Reporter

Part:BBa_K1468004

Designed by: Pedro Luis Dorado Morales   Group: iGEM14_Valencia_Biocampus   (2014-10-03)

pTet + gene encoding LacZ


Contents

Description
Usage and Biology, Universality
Innovative point
Doubts and FAQs
Further reading and additional information

Description

This part consists of the LacZ coding sequence of E. coli, placed under the control of the tetracycline-responsive promoter pTet -constitutively "on" promoter which is repressed by TetR (repression inhibited by the addition of tetracycline or an analog of this molecule, as the doxycyline).


Usage and Biology, Universality

The regulatory elements that control tetracycline resistance in E. coli were converted into highly specific transcription regulation systems that can work in a wide variety of eukaryotic cells. Despite its intriguing properties, its use is limited, particularly in transgenic animals, because of its relatively inefficient inducibility by doxycycline in some organs, its instability, and its residual affinity to tetO in absence of Dox, leading to elevated background activities of the target promoter.

To remove these limitations, tTA DNA (one tetracycline respressor -TetR- mutant) have been mutagenized and selected in Sacharomyces cerevisiae for obtaining mutants with reduced basal activity and increased Dox sensitivity.

Summing up, BBa_K1468004 can be used in a wide variety of prokariotic and eukariotic cells (see Further reading and additional information).

Some considerations about the biobrick:

- The LacZ encoding gene sequence owns to Escherichia coli, fact that ensures its correct operation in a group of closed related species but not to the wide range of microorganisms. Some codon usage modification must be carried out for non-related organisms.

- This system is not limited to the application specified in the Valencia Biocampus 2014 iGEM project [1]. According with the usage of interest, we can change the lacZ gene for other sequence we want to express in a concret chassis or process.

- There are some limitations related to the plasmid, where the construction is cloned. I mean, the backbones Ori is not universal, so it would be necessary to check that if Ori is compatible with the organism we want to transform.

Innovative point

The BBa_K1468004 part presents a deep characterization in a range of E. coli strains (see experience [2]).

Doubts and FAQs

For any question, send an e-mail to the address shown below and we will reply you as soon as possible.

vlc.biocampus.igem@gmail.com

Further reading and additional information

Kauth, C.W. et al. The merozoite surface protein 1 complex of human malaria parasite Plasmodium falciparum: interactions and arrangements of subunits. The Journal of Biological Chemistry (2003) 20, 22257-22264

Pan, W. et al. Vaccine candidate MSP-1 from Plasmodium falciparum: a redesigned 4917 bp polynucleotide enables synthesis and isolation of full-length protein from Escherichia coli and mammalian cells. Nucleic Acids Research (1999) 5, 1094-1103

Welman, A. et al. Tetracycline regulated systems in functional oncogenomics. Translational Oncogenomics (2007) 2, 17–33

Baron, U. & Bujard, H. Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances. Methods in Enzymology (2000) 327, 401-421

Furth, P.A. et al. Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proceedings of the National Academy of Sciences (1994) 27, 9302-9306.

Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proceedings of the National Academy of Sciences (1992) 15, 5547-5551

Burghaus, P.A. et al. Analysis of recombinant merozoite surface protein-1 of Plasmodium falciparum expressed in mammalian cells. Molecular and Biochemical Parasitology (1999) 30, 171-183

Zhou, X. et al. Optimization of the Tet-On system for regulated gene expression through viral evolution. Gene Therapy (2006) 13, 1382–1390

Urlinger, S. et al. Exploring the sequence space for tetracyline-dependent transcriptional activators: Novel mutations yield expanded range and sensitivity. Genetics (2000) 97, 7963-7968

Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Cell biology (1992) 89, 5547-5551


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//cds/enzyme
//cds/reporter
//chassis/multihost
//chassis/prokaryote
//chassis/prokaryote/ecoli
//classic/regulatory/other
//classic/reporter
//classic/reporter/constitutive
//function/reporter
//function/reporter/color
//regulation/constitutive
//regulation/negative
//terminator
Parameters
chassisE.coli strains: DH5-alpha, XL1 Blue, JM109, DH10B, HB101, BL21 (DE3)
colorBlue
controlTetR repressible promoter (Promoter constitutively on)
negative_regulatorsTetR
outputX-Gal catalysis product (Blue precipitate)
positive_regulatorsTetR repression inhibited by tetracycline or an analog (doxycycline, i.e.).
proteinLacZ